The goal of the work described in these studies is to elucidate how critical extracellular and intracellular domains control signaling by notch receptors. The extracellular domains of all notches contain three iterated LIN12 repeats, which maintain notch receptors in their resting state prior to ligand binding. Ligand-induced proteolysis releases the intracellular portion of notch (ICN) and allow it to translocate to the nucleus, where its RAM and ankyrin-repeat domains interact with two additional factors, RBP-JK and mastermind (mami 1) to form a transcriptionally active ternary complex. The specific aims of this research plan, which will provide important new insights into the structure and function of the LIN12 and ankyrin repeat domains of notch, are to: 1. Determine how LIN12 modules prevent ligand-independent activation of notch receptors. (a) Determine high-resolution solution structures of key modules and module pairs from a Notch LIN12 domain, analyzing the structural interactions of these modules with each other and with neighboring domains. (b) Identify contacts between the LIN12 domain and the NTM extracellular stub that maintain the integrity of the Notch heterodimer, and (c) Test the functional importance of specific intermodular and LIN12-NTM contacts. 2. Determine the essential domains and residues required for assembly and function of thenotch/RBP-Jkappa/maml1 signaling complex. (a) Determine the minimal protein domains required for reconstitution of an ICN-mam11-RBP-Jkappa--DNA complex. (b) Identify contacts that contribute to energetic stabilization of ICN complexes and determine how RBP and RAMANK cooperate to recruit mamli to form a ternary complex on DNA. (c) Test the function of specific contacts responsible for stabilizing the ternary DNA-binding complex.